Artemisinin combination therapies (ACTs) are the mainstay of treatment for Plasmodium falciparum. However, slow clearance of parasites from the blood following treatment with ACTs in some SE Asian countries has raised fears of impending resistance. The central goals of this renewal application are to identify parasite genes that underlie slow clearance rate (CR) following treatment with artemisinin (ART), to understand the evolution of this trait, and to probe the underlying mechanisms using transfection. To identify markers for slow CR, we will work with malaria parasites from the Thai-Burma border, because patients in this region show a wide range of CR following ART treatment, and we have shown that the most (58%) of the variation in CR can be explained by parasite genetic factors. We will use 650 finger-prick parasite DNA samples collected since 2007 for this analysis. These samples are genetically unique (from genotyping 96 SNPs), contain single malaria genotypes and have robust (6 hourly) measures of parasite clearance rate. We will genotype these samples at 16,875 polymorphic single nucleotide polymorphisms (SNPs) using a Nimblegen microarray specifically designed for SE Asian parasites, impute additional SNPs from a Thai reference parasite population for which 101 whole genome sequences are available, and identify the genes that underlie CR using a genome wide association study (GWAS). To confirm involvement of these loci, we will genotype candidate genes identified in 12 independent parasite populations from six SE Asian countries, with 10 year longitudinal sampling from Thailand and Cambodia. These data will allow verification of associations from our GWAS, determine the distribution of causative alleles, track changes in allele frequency of candidate loci over time, and identify numbers of independent origins of this trait. There are currently no good phenotypic assays of the slow CR trait for use in the laboratory. We will evaluate the utility of two promising measures, a quantitative recrudescence assay and a flow cytometry-based growth assay, which effectively differentiate between ART resistant parasites selected in the laboratory and their sensitive progenitors. This will be done using panels of slow and fast clearing parasites from the Thai-Burma border, for which cryopreserved stocks are available. Finally, to determine causality and investigate the underlying mechanisms of slow CR, we will manipulate expression of candidate genes and examine how this alters surrogate in vitro measures associated with the slow CR phenotype. Effective partner drugs are critical for maintaining effective treatment using ACTs. We will also use transfection to examine candidate markers for resistance to partner drugs identified from a GWAS study conducted during the previous grant period. PUBLIC HEALTH RELEVANCE: Artemisinin combination therapy (ACT) is the main treatment for Plasmodium falciparum malaria: the success of this treatment has rolled back malaria and renewed interest in malaria elimination. Slow clearance of parasites following ACT treatment of patients in SE Asia has lead to concern about the spread of resistance. This proposal aims to identify the genes that underlie slow clearance to better understand the evolution and biochemical basis of his trait.